Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

Immunocytochemical study of the intracellular localization of M protein of vesicular stomatitis virus

Summary The purpose of this paper is to describe the immunocytochemical localization of M protein of vesicular stomatitis virus (VSV) in infected cells. Vero cells, MDBK cells, Swiss 3T3 cells, and BHK cells were examined at various times after infection. For immunofluorescent staining, the cells were fixed with PLP fixative and then treated with 0.05% Triton X-100 before incubation with antibodies. Three hours after infection, M protein exhibited diffuse immunostaining throughout the cytoplasm and later accumulated along the cell membrane. The localization of M protein differed from the granular localization of the nucleocapsid N protein of VSV in the cytoplasm. For electron microscopy, the cells were fixed first in a mixture of 2% paraformaldehyde and 0.05% glutaraldehyde and then with PLP fixative, this being followed by treatment with 0.05% saponin. They were then immunostained using the immunoperoxidase method. The M protein was found to be distributed throughout the cytoplasm and later under the cell membrane, especially at virus budding sites. We also used postembedding immunostaining and freeze-fracture immunostaining to avoid the translocation of M protein caused by the detergent treatment. These techniques confirmed our previous results. Our findings are consistent with the view that the M protein of VSV is synthesized on free ribosomes and is then associated with the cell membrane where viral assembly may occur.S. Ohno was a visiting fellow from the Fogarty International Center at the National Institutes of Health, USA, from September 1981 to August 1983, while some parts of this work were in progress.     

The usefulness of alkaline solutions for clearing the uterus and staining implantation sites in rats

The alkaline solution method for detecting uterine implantation sites (metrial glands or spots after delivery), which indicate the position of the embryo, was compared with conventional methods using Turnbull blue (Salewski's technique) and benzyl benzoate (Orsini's technique) in rats. When rat uteri were immersed directly in 2% sodium hydroxide [NaOH], potassium hydroxide [KOH] solution or one of several other alkaline solutions for one hour, the uteri were cleared. Then the metrial glands could be observed without further staining, and the spots after delivery with further staining. After alkaline treatment, unstained metrial glands appeared as yellowish-white degenerated masses and stained spots after delivery appeared as yellowish-brown patches covered with yellowish-white lumps. These yellowish-brown or yellowish-white sites were probably stained by the iron in hemosiderin deposits in the placental scar or in a vestige of the metrial glands left over from pregnancy. Formalin-fixed uteri were not cleared by immersion in a high concentration of NaOH, KOH or other alkaline solutions, but the spots after delivery were stained by this treatment. Turnbull blue did not stain the metrial glands, which contain no iron. Benzyl benzoate cleared fixed or unfixed uteri and allowed observation of the metrial glands and spots after delivery although the entire process required a few days and the specimens became hard and shriveled. The best and most convenient solution for clearing the rat uterus and for staining implantation sites was 2% NaOH or KOH.     

Immunohistochemical Detection of Sialyl LeX Antigen on Mucosal Langerhans Cells of Human Oral Mucosa following Neuraminidase Pretreatment

Histochemical assessment of selected carbohydrate sequences on Langerhans cells of human oral mucosa was made by combined use of enzyme digestion and immunostain-ing with monoclonal antibodies against specific carbohydrate structures. In both frozen sections and epithelial sheets without the enzyme pretreatment, mucosal Langerhans cells, identified by positive staining with anti-CD1a and HLA-DR antibodies, did not express any carbohydrate antigens on their surface. In contrast, following neuraminidase pretreatment of both types of material, the fucosylated type 2 chain (Le^X) became detectable on Langerhans cells, indicating that sialic acid is the terminal residue of this sequence. Other enzymes were ineffective in this apparent unmasking, and the staining patterns of the other related carbohydrate sequences (Le^y. Le^a, Le^b) remained unaffected by pretreatment with any of the enzymes used. These findings suggest that the mucosal Langerhans cells possess a unique carbohydrate chain, the sialyl fucosylated type 2 sequence (sialyl Le^X antigen).     

Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation

Production of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa-14, a gene related to surfactin production cloned at our laboratory from a wild-type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37 degrees C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30 degrees C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4-5 times more efficient than in SMF. (c) 1995 John Wiley & Sons, Inc.     

Comparative reproductive patterns in culture of differentGigartina subgenusMastocarpus andPetrocelis populations from northern Japan

Samples of theGigartina pacifica-ochotensis complex were collected at 21 localities around Hokkaido and northern Honshu. The carpospore and blade tip cultures showed 3 reproductive patterns. (1) 237 (86.8%) of the 273 cultured isolates derived from single plants have a direct type of life history. (2) 29 (10.6%) isolates exhibited a heteromorphic type with the alternation of foliose gametophytes and crustose tetrasporophytes. (3) 7 (2.6%) isolates showed a mixed pattern in which carposporelings developed intoPetrocelis-like crusts, basal discs with uprightGigartina blades, or chimera-like discs with compositePetrocelis-Gigartina anatomy. CulturedGigartina blades derived from bothG. pacifica andG. ochotensis were similar in morphology. In 18 cultures from 5 localitiesPetrocelis tetraspores developed into dioeciousGigartina gametophytes. A single tetrasporeling grew into aGigartina plant that reproduced directly. In hybridization experiments with 8 male and 14 female isolates from 4 localities on Hokkaido 85 (78.0%) of 109 were positive. On the basis of these and earlier studies it is concluded that a single species is present in northern Japan:G. pacifica Kjellm. has priority overG. ochotensis (Rupr.) Rupr. ex Yendo.